PROJECT ABSTRACT The degeneration of cartilage at load-bearing joints causes considerable musculoskeletal morbidity and is a major healthcare burden in the United States. While several treatment options exist, including autologous chondrocyte transplantation and implantation of tissue-engineered substitutes, they are expensive and of limited efficacy. The goal of the proposed research is to develop a novel method to improve the therapeutic outcomes of microfracture (MF) by engineering a treatment modality that delivers low-intensity ultrasound (LIUS; < 50 mW/cm2) with chondroinductive and chondroprotective properties to the defect site. Two specific aims will be undertaken to accomplish the project goal: (1) develop a theoretical basis for LIUS therapy and (2) demonstrate efficacy of LIUS in improving MF outcomes in a rabbit model of chondral injury. This work goes beyond existing empirical approaches reported in the literature by proposing to identify the in-vivo resonance frequency range where the bioeffects of LIUS are maximized and link the macroscopic settings of transducer frequency, power, and exposure times to repair outcomes in order to reduce the variability in clinical outcomes and optimize efficacy. This R21 proposal integrates and builds on the project team's innovative finding that the bioeffects of ultrasound (US) are frequency-dependent and that the cell response to US is maximized at the resonant frequency and the biological finding that in-vitro LIUS at the resonant frequency aids the proliferation of mesenchymal stem cells (MSCs); promotes chondrogenic differentiation of MSCs; and maintains the chondrocytic phenotype by upregulating SOX9, the key transcription factor in chondrogenesis. The project team's aggregate in-vitro preliminary data suggest that the use of LIUS will significantly improve cartilage regeneration procedures that seek to recruit MSCs and induce lineage-specific conversion without the use of growth factors that pose common side effects such as hypertrophy and fibrosis. These data led to this project's central hypothesis that LIUS regimens can benefit cartilage repair therapies in three ways: (1) LIUS can enhance the migration of progenitor cells, (2) LIUS can enhance in situ chondrogenic differentiation of MSCs, and (3) continuing LIUS stimulation of chondrocytes improves the formation of hyaline cartilage. This hypothesis- driven research promises to result in a more fundamental understanding of the therapeutic value of LIUS, which is expected to ultimately lead to the development of a LIUS-based regimen and delivery system capable of generating a stable hyaline cartilage phenotype via minimally invasive MF procedures. Such progress would, in turn, directly address the needs of 46 million Americans who suffer from osteoarthritis.